Functional, Aromatic, and Fluorinated Monothiosemicarbazones: Investigations into Their Structures and Activity toward the Gallium-68 Incorporation by Microwave Irradiation

We report on the synthesis and spectroscopic characterization of a new series of coordinating monothiosemicarbazones incorporating aromatic backbones, featuring O/N/S donor centers monosubstituted with different aliphatic, aromatic, fluorinated, and amine-functionalized groups at their N centers. Their ability to bind metal ions such as Zn(II) and Ga(III) was explored, and the formation of two different coordination isomers of the Zn(II) complex was demonstrated by X-ray diffraction studies using synchrotron radiation. These studies showed the planar geometry for the coordinated mono(thiosemicarbazone) ligand and that the metal center can adopt either a heavily distorted tetrahedral Zn center (placed in an N/S/S/N environment, with CN = 4) or a pseudo-octahedral geometry, where the Zn(II) center is in the O/N/S/S/N/O environment, and CN = 6. Furthermore, 2-(4,5-dimethyl-2-thiazolyl)-3,5-diphenyl-2H-tetrazolium bromide (MTT) assays and cellular imaging in living cells were subsequently performed in two different cancer cell lines: PC-3 (a standard cell line derived from a bone metastasis of a stage IV prostate cancer) and EMT6 (a commercial murine mammary carcinoma cell line). The radiolabeling of new functional and aromatic monothiosemicarbazones with either gallium-68 (under pH control) or fluorine-18 is discussed. The potential of this class of compounds to act as synthetic scaffolds for molecular imaging agents of relevance to positron emission tomography was evaluated in vitro, and the cellular uptake of a simultaneously fluorinated and [68Ga]-labeled mono(thiosemicarbazone) was investigated and is reported here.

1.General experimental methods 2.Synthetic procedures with alternative routes and selected analytical data 3.Optical spectroscopy of selected monothiosemicarbazones and kinetic stability tests 4.Cellular imaging and viability assays 5.Radiochemistry assays 6.Cellular uptake of the Gallium-68 radiolabeled compound 4a 7. Experiments for the formation of 'cold' Gallium (III) complexes 8.Additional mass spectrometry data for key compounds and intermediates 9.Infrared spectroscopy for representative compounds 10.Selected X-Ray Crystallographic Data 11.X-ray diffraction data for 2a, 3a, 4a, 4b and 4c 12. CCDC Deposition Information 13.Estimation of logP 14.Gas Phase DFT Calculations

Alternative methods for the preparation of mono(4-ethyl-thiosemicarbazone)acenaphthenequinone (4f)
The ligand was obtained as reported in the literature from a 1:1 molar ratio of acenaphthenequinone and thiosemicarbazide in absolute ethanol. Hereby, the reaction were carried out using acenaphthenequinone (0.5 g, 2.74mmol) and 4-ethyl-3-thiosemicarbazide (0.358 g, 3.00 mmol), which were suspended in absolute ethanol (15ml) and heated under reflux for 2 hours. The solid was isolated by filtration whilst hot then re-suspended in hot methanol (10ml) and stirred for 15 minutes before filtering and washing with further methanol. The resulting solid was then dried under vacuum purity checked using HPLC under various conditions. Yield = 0.659 g, 2.329 mmol, 85%.
Synthesis of mono (4-allyl-3-thiosemicarbazone) acenaphthenequinone (4g) Compound 4g was prepared as a result of the reaction between acenaphthenequinone (0.1 g, 0.5489 mMol) and 4-allyl-3-thiosemicarbazone (0.0864 g, 0.6587 mMol) in a 1:2 ratio. Both compounds were dissolved in 40ml of ethanol and a few drops of glacial acetic acid were added. The reaction was refluxed at a maximum temperature of 100 o C for 4 hours. Following the removal of methanol with evaporation, a dark yellow solid was isolated by filtration and washing with diethyl ether.

Confocal fluorescence microscopy experiments
PC3 cells were cultured under normoxic conditions as previously described, and then seeded in a glass bottom dish at least 48 h prior to the microscopy experiment (10,0000 cell per well plate) and incubated at 37 ºC. Control fluorescence images were recorded before the addition of compound. For confocal microscopy, the compound in a DMSO: RPMI/SFM 0.5:99.5 solution mixture (50 μM), was loaded into the wells and cells were allowed to incubate for 15 min, at 37 °C. They were then carefully washed with PBS and then it was replaced by RPMI/SFM to remove the non-internalised fluorescent dispersion prior to fluorescence imaging.

MTT assays
Cells (5-7×10 3 cells per well) were seeded on a sterile 96-well plate and incubated for 48 h to adhere. All the TSCa mono(substituted) ligands were subsequently loaded at different concentration into wells and cultured for another 48 h. Subsequently, cells were washed three times with PBS and 3-(4, 5-dimethylthiazol-2-yl)-2, 5diphenyltetrazolium bromide (MTT) was added (0.5 mg/ mL, 90% serum-free medium (SFM)) + 10% PBS followed by a two-hour incubation. After aspiration, 100 μL of DMSO was added and the 96-well plates were read by an ELISA plate reader, Molecular Devices Versa Max (BN02877). The absorption wavelength was at 570 nm and 630 nm wavelength was used as a reference. For establishing the correct protocols, a library of known compounds with similar solubility and which also carries the N/S motifs were analysed.

General synthetic procedure for the synthesis of the Gallium-68 acenaphthenequinone thiosemicarbazones complexes
A SnO2-based column matrix 68 Ge/ 68 Ga generator was used to elute 10 mL of 0.6 M HCl, ca. 4 mCi of 68 Ga, which was trapped on a strata x-c 33 μm Polymeric Strong Cation Cartridge from Phenomenex and eluted with 700 μL of 0.02M HCl (98% THF). This was subsequently dried for 7-10 min under a nitrogen stream at 95°C. Next 30 μL of the monosubstituted compound in dry DMSO (2mg/mL) was added along with 0.6 mL of injectable MeOH and the pH adjusted to range between 5-7. This was heated under microwave radiation at 95 °C for 30 min. Analysis by reverse-phase HPLC (Method B) gave two different retention times for each compound which in comparison with the HPLC trace of the precursors suggest that this could be isomers of the compound. Remains of the [ 68 Ga]GaCl3 were indicate that radiolabelling of the mono(substituted) ligands had not gone to completion. It was found that if reactions were carried out at pH ca 1-4 only very limited conversions were achieved, either by microwave irradiation or by conventional heating, even under prolonged reaction times (60 min -120 min).

Cellular uptake of the Gallium-68 radiolabeled compound 4a
PC-3 and EMT-6 cells (3·10 3 cells) were seeded in 6-well plates and incubated in normoxia and hypoxia environments as described before. After treatments, plates were aspirated and washed twice with warm PBS buffer. Each well plate was then loaded with 1000 μL of Gallium-68 complex in DMSO:PBS solution mixture (0.5:95.5) (3 MBq/ mL) and incubated for 1 h. After incubation, the reaction was stopped by washing wells with ice-cold PBS buffer twice, followed by addition of 1 mL of ice-cold, 0.1 % Triton X-100 and 0.1 M NaOH Lysates. A homogenous mixture was obtained by blending the components with up/down pipetting. 800 μL of each dissolved cell was then transferred and capped into counting tubes for gamma counting. The stock of the gallium-68 complex solution was aliquoted in 3 of 10μL dose and placed to the counting tubes as standards.
The intracellular radioactivity was immediately counted using an LKB Wallac 1282 Compugamma Laboratory gamma counter (PerkinElmer, USA). Lastly, protein concentration determination was carried out by BCA assays. The cells were counted at a Sunrise absorbance reader (Tecan Trading AG, Switzerland). This normalisation of decay-corrected radioactivity counts per minute (CPM) to protein concentration, was required in order to give a measure of radiotracer uptake as % ID/mg of protein= CPM in 1 mL/ (standard in mL · protein concentration in mg) · 100 %. All the obtained results were analysed through the scientific 2D graphics and statistics software GraphPad Prism (GraphPad Software, California).

Investigations into the formation and nature of 'cold' Gallium (III) complexes
Formation of a Ga(III) (F-benzyl thiosemicarbazonate) acenaphthenequinone complex using a microwave protocol either directly, from the free ligand 4a or through the intermediate formation of the Zn(II) precursor (denoted 4a-Zn) was performed. However only the formation of an intractable mixture of species was observed and the desired compound with proposed structure as shown below (denoted 6a, or 4a-Ga) could not be isolated in a pure form by semi-prep HPLC. 19 F NMR spectroscopy 19 F{ 1 H} NMR (DMSO-d6, 25 ºC): showed broad resonance at -69 ppm and -120 ppm, additionally to the singlet peak characteristic to a 4a-type fluorine resonance (assignable to the free ligand 4a or Zn-bound 4a) at -118 ppm.

Pathway to the anhydrous synthesis of Ga-4f using NEt3
In a glove box, 2 equivalent of compound 4f (0.07 g, 0.25 mmol) was suspended in anhydrous THF (10 ml) and 1 equivalent of anhydrous GaCl3 (0.022 g, 0.125 mmol) was added to the suspension. An instant colour change from yellow to red was observed upon the addition of 0.025 ml triethylamine. The mixture was then stirred for 24 h at room temperature under argon. Pentane (15 ml) was then added to the solution and the mixture was placed in the freezer overnight and a yellow solid crashed out. This solid (fraction A) was removed by filtration. The filtrate had solvent removed by rotary evaporation to leave a brown solid (fraction B). Both fractions were analysed by mass spectrometry which indicated formation of mixtures of Ga-containing products as well as free ligand.

Alternative procedure for synthesis of Ga-4f under anhydrous conditions
Compound 4g (mono Et-TSC (0.07 g, 0.25 mmol) was suspended in anhydrous THF (10 ml) and 1 molar equivalent of anhydrous GaCl3 (0.022 g, 0.125 mmol) was added to the suspension. The mixture was stirred for 24 h at room temperature under argon. Pentane (15 ml) was then added to the solution and the mixture was placed in the freezer overnight and a yellow solid crashed out. This solid (fraction A) was removed by filtration. The filtrate had solvent removed by rotary evaporation to leave a brown solid (fraction B).

Pathway to the anhydrous synthesis of Ga-4g using NEt3
In a glove box, the ligand 4g (mono Allyl-TSC, 0.07g, 0.25 mmol) was suspended in anhydrous THF (10 ml), 1 equivalent of anhydrous GaCl3 (0.022g, 0.125 mmol) was added carefully to the suspension observing a 2:1 ratio of ligand to [Ga]. Then to this mixture, 0.025 ml triethylamine was added and an instant colour change was observed from yellow to a darker orange. The mixture was stirred for 24 h at room temperature under argon. Pentane (15 ml) was then added to the solution and subsequently the mixture was placed in the freezer overnight and an orange solid crashed out. This solid (fraction A) was removed by filtration. The filtrate had solvent removed by rotary evaporation to leave an orange solid (fraction B). Both fractions were analysed by Mass Spectrometry which showed that whilst it is possible to isolate the desired species, additional gallium containing fragments may also form from this reaction, even under the carefully controlled anhydrous conditions utilised.   solvents, d6-DMSO and H2O). (fraction B, made from the anhydrous synthesis reaction). Data suggests that only traces of the desired gallium compounds have been isolated.

Alternative procedure towards the formation of Ga-4g
Compound 4g, mono Allyl-TSC (0.07 g, 0.25 mmol, 2 molar equivalent) was suspended in THF (10 ml). 1 equivalent of anhydrous GaCl3 (0.022 g, 0.125 mmol) was dissolved in degassed water (0.5 ml), then THF (2 ml) was added. The GaCl3 solution was added dropwise to the suspension of mono Allyl-TSC. The mixture was stirred for 24 h at room temperature. Pentane (15 ml) was then added to the solution and the mixture was placed in the freezer overnight and an orange solid crashed out. This solid (fraction A) was removed by filtration. The filtrate had solvent removed by rotary evaporation to leave an orange solid (fraction B).

Reaction between excess aqueous [Ga(III)] and 4g
A solution of aqueous GaCl3 (0.7 mol dm -3 ) was prepared in degassed water. (Note: It was assumed this solution then contained a mixture of GaCl3 and Ga(OH)3, either as monomers or as oligomers, as well as other solvated Ga(III) species). The ligand 4g (0.049g, 0.17mmol) was dissolved in 30 ml of THF in a Schlenk flask under N2. Then, 1 ml of the [Ga] stock solution was added by syringe to the THF ligand solution. The mixture was left to stir for 24 hours at the room temperature. Over that period the solution became gradually darker, before becoming dark orange. To this solution, 5 mL of pentane was added under stirring and the resulting slurry was transferred to a round bottom flask. The solution was left in a freezer overnight where a dark orange solid crashed out. This solid was removed by filtration giving rise to a solid denoted 4g-Ga (fraction A), which was analysed by ESI-MS+. From the filtrate fraction, the solvent was removed under reduced pressure and the second fraction (denoted 4g-Ga fraction B) was also analysed by ESI+. In both cases the 1 H NMR spectroscopy showed formation of complex mixtures of products and free ligand. A ESI+ MS analysis was performed on fraction A of 4g-Ga. A close inspection showed one peak with m/z = 296.1246 corresponded to the free ligand, as well as un-assignable [Ga] containing peaks at higher m/z. The ES+ MS of fraction 4g-Ga B also indicated the presence of fragments corresponding to the free ligand as well as fragments with high m/z: e.g. one peak of interest, m/z = 729.3620 was assignable to a gallium-ligand complex of type [GaL2Cl2+H + ] where the two chlorines seem to remain associated with the complex ion in mass spectrometry. The compound 4h mono Ph-TSC (0.08 g, 0.25 mmol, 2 equivalent) was suspended in THF (10 ml). 1 equivalent of anhydrous GaCl3 (0.022 g, 0.125 mmol) was dissolved in degassed water (0.5 ml), then THF (2 ml) was added. The GaCl3 solution was added dropwise to the suspension of mono Ph-TSC. The mixture was stirred for 24 h at room temperature. Pentane (15 ml) was then added to the solution and the mixture was placed in the freezer overnight and a dark red solid crashed out. This solid (fraction A) was removed by filtration. The filtrate had solvent removed by rotary evaporation to leave a red solid (fraction B). In a glove box, the ligand 4h (mono Ph-TSC, 0.08 g, 0.25 mmol) was suspended in anhydrous THF (10 ml) and treated with anhydrous GaCl3 (0.022 g, 0.125 mmol) which was carefully added to the suspension, aiming to closely observe the 2: 1 molar ratio of 4f to GaCl3 added. An instant colour change was observed upon the addition of 0.025 ml triethylamine to this mixture. The resulting mixture was stirred for 24 h at room temperature under argon. Pentane (15 ml) was then added to the solution and the mixture was placed in the freezer overnight and a dark red solid crashed out. This solid (fraction A) was removed by filtration, and the mass spectrometry analysis proved unconclusive. The filtrate had solvent removed by rotary evaporation to leave a red solid (fraction B). Detailed analysis by Mass spectrometry revealed recovery of free ligand 4h, as well as the presence of the desired Ga-4f species in Fraction B.
To this suspension, anhydrous GaCl3 (0.022 g, 0.125 mmol) was added (1 molar equivalent). The mixture was stirred for 24 h at room temperature under argon. Pentane (15 ml) was then added to the solution and the mixture was placed in the freezer overnight and a dark red solid crashed out. This solid (fraction A) was removed by filtration. The filtrate had solvent removed by rotary evaporation to leave a red solid (fraction B).

Alternative method for the reaction between GaCl3 and Compound 4h
Compound 4h (0.051g, 0.15mmol) was added to a solution of dry THF (20 mL) containing GaCl3 whilst the reaction vessel was kept under a nitrogen. (Note: Anhydrous GaCl3 (0.0135g, 0.077mmol) was weighed out and added to a round bottom flask provided with a septum in a glove box). The reaction mixture was left to stir for 24 hours under N2, then a small amount of pentane was added to the solution and the mixture was placed in the freezer overnight. A red solid crashed out and this solid (denoted 4h-Ga fraction A) was removed by filtration. The filtrate was concentrated by rotary evaporation to generate a red oily solid residue (denoted 4h-Ga B). 1 H NMR analysis in d 6 -DMSO proved inconclusive with formation of protonated ligand species being observed. The HPLC was dominated by the free ligand species, indicating decomposition in wet solvents. The Mass spectrometry (ESI+) of fraction 4h-Ga A did not show expected molecular ion peak centred at m/z 753 region. Instead, a peak centred at m/z = 354.1161, representing the free ligand associated with Na, was observed together with peaks with higher m/z. The fraction denoted 4h-Ga B was also analysed by ES+ MS and a small peak for the m/z = 753.2173 was found, most closely assignable to a [Ga(HL)(L)+Na] fragment, where HL corresponds to the protonated ligand (4h). The complex Zn-4f (Zn(EtTSC)2, 0.06g, 0.10 mmol) was suspended in dried MeOH (20 ml), and then added dropwise to a flask containing neat and anhydrous GaCl3 (0.18 g, 0.125 mmol). The mixture was then heated to 65 ℃ over 5 h under argon. The reaction mixture was allowed to reach room temperature, and the slurry was subsequently concentrated under reduced pressure to yield a red oil. To this, a small amount of Et2O (10 mL) was then added and a red precipitate formed. The precipitate (fraction A) was removed by filtration. The filtrate had solvent removed by rotary evaporation to leave an orange solid (fraction B). Both fractions were analysed by mass spectrometry, to show the presence of the Zn-4f precursor, additionally to the expected Ga-4f compound.
The reaction mixture was then heated to reflux over 5 h. Then, the reaction mixture was allowed to reach room temperature, and the resulting slurry concentrated under reduced pressure to yield a red oil. To this, a small amount (2 mL) of Et2O was added. This resulted in the formation of a red precipitate which was isolated by filtration (18.2 mg). The 1 H NMR (d 6 -DMSO) showed the presence of the free ligand additionally to a complex mixture of species. By ESI+ main fragment is assignable to a species containing Ga ion as monocoordinated to one 4g ligand and 2 OH groups, [C16H14GaN3O3S+Na] with m/z centred at 419.3482, and the presence of the desired Ga-4f species is also indicated by the higher m/z peaks assignable to [M]+ and [M+H+Cl]+. ESI+ and ESI-mass spectrometry support the formation of the GaL2X2 as well as GaLX2 species, however the formation of the free ligand 4g was also observed. Additionally, the presence of the 4g-Zn starting material cannot be fully discounted by 1 H NMR and ESI+ MS.

Figure S95
Mass Spectrometry (ESI+) of the resulting product mixture from the treatment of 4g-Zn compound with excess Ga(III) recorded after the transmetallation reaction and repeated under a number of injection conditions.

Figure S96
Mass Spectrometry (ESI-) of the resulting product of 4g-Zn with excess Ga(II) recorded after the transmetallation reaction. Figure S97. The 1H NMR spectroscopy in d 6 -DMSO for the transmetallation reactiona comparison of the 1 H NMR for the free ligand 4g (top spectrum), complex 4g-Zn (middle spectrum) and the products of the Zn(II) to Ga(III) transmetallation reaction (bottom spectrum; free ligand and 4g-Ga mixture, see Scheme S8).

Synthesis of Ga-(Et-TSC)2 by transmetallation reaction (MeOH)
The complex 4f-Zn (Zn(EtTSC)2, 0.06g, 0.10 mmol, 1 molar equivalent) was suspended in anhydrous MeOH (20 ml) , added dropwise to 10 equivalent of anhydrous GaCl3 (0.18 g, 1 mmol) and refluxed at 65 ℃ for 5 h under argon. The reaction mixture was allowed to reach room temperature, and the slurry was concentrated under reduced pressure to yield a red oil. Et2O was then added until a red precipitate formed. The precipitate (fraction A) was removed by filtration. The filtrate had solvent removed by rotary evaporation to leave an orange solid (fraction B) which was analysed by mass spectrometry.

Alternative synthetic approach towards Ga-(Et-TSC)2 by transmetallation reaction (THF)
The compound 4f, (Zn(EtTSC)2,0.06g, 0.10 mmol, 1 equivalent) was suspended in anhydrous THF (20 ml) , added dropwise to 10 equivalent of anhydrous GaCl3 (0.18 g, 1 mmol), refluxed at 65 ℃ for 5 h under argon. The reaction mixture was allowed to reach room temperature, and the slurry was concentrated under reduced pressure to yield a red oil. Et2O was then added until a red precipitate formed. The precipitate (fraction A) was S75 removed by filtration. The filtrate had solvent removed by rotary evaporation to leave an orange solid (fraction B).

DFT Geometry optimizations of 4f-Zn and 4f-Ga complexes
Zn-1 Zn-2 Alternative view Zn-1 Alternative view Zn-2  Table S9. Optimised energies for the two isomers of the Zn(II) complex of ligand 4f.

DFT investigations into the geometries of the Ga(III) complex isomers of ligand 4f
Figure S117. Optimised geometries of the two isomers of the monocationic Ga(III) complex ion of ligand 4f. The Clcounterion was not included in the model.